Human prion diseases are fatal neurodegenerative disorders that have familial, sporadic and transmissible etiologies, yet no therapeutics exist. Our knowledge of prion diseases has centered on the infectious scrapie prion protein (PrPSc) interacting and converting the normal cellular prion protein (PrPC) into the pathogenic conformer, PrPC?PrPSc. Experiments in cell culture systems have demonstrated that PrPC?PrPSc conversion occurs near the cell membrane followed by PrPSc trafficking into the cell. Yet the precise cellular localization after internalization is controversial as different antibodies and cell culture methods yielded variable results. Further, different prion strains will likely complicate this matter, as each strain has unique biochemistry and neuropathology characteristics. Moreover, studies in transgenic mice, cell culture systems and in vitro conversion assays have strongly argued that the interaction between PrPC and PrPSc is not entirely sufficient for prion propagation. These observations suggest additional modifiers are required. Thus, two major goals of this proposal are to characterize the cellular phenotypes of mammalian prion strains and identify factors that guide prion propagation. To ascertain the cellular localization of prion strains, in Aim 1, I will engineer cells to express Mouse PrPC fused to a small tetracysteine (TC) peptide tag, which binds a fluorescein derivate known as FlAsH. Prion infected cells will be pulse-chased with FlAsH to demonstrate de novo PrPC?PrPSc conversion and reveal the inter/intracellular localization of prion strains. This approach has advantages over traditional immunohistochemical techniques, as FlAsH is rapid and can only label PrPC, thus imparting a time-stamp for conversion. Further, in Aim 2, I will investigate how human prions propagate in mammalian cells. I will engineer cells to express Human PrPC fused to a TC tag. Brain extracts from patients who died of a prion disease will be applied to cells and FlAsH pulse-chased to detect PrPC?PrPSc conversion. In addition, comparative studies between FlAsH labelled mouse and human prions will be done to tease out a general mechanism for mammalian prion trafficking in a cell. In Aim 3, the clustered regularly interspaced short palindromic repeats (CRISPR) system will be used to globally screen genes that modulate FlAsH labelled mouse and human prions in cells. The results of this screen may reveal modifiers of mammalian prion propagation. Collectively, determining the cellular organelles prions harbor during pathogenesis and identifying the factors that propagate prions in cells, may lead to therapeutics to Human prion diseases. Given that Alzheimer's and Parkinson's diseases have prion-like properties of infection, the impact of this study may encompass other neurodegenerative disorders.